Compounds and Methods for Modulating an Immune Response

Abstract

The present invention relates to the identification of proteins which bind the dendritic cell marker known as Clec9A. The present invention provides new compounds for targeting therapeutic agents such as antigens to dendritic cells. Also provided are methods of modulating a humoral and/or T cell mediated immune response to the antigen, methods of delivery of a cytotoxic agent to dendritic cells thereof involved in diseased states, methods of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, and methods of modulating antigen recognition, processing and/or presentation, as well as immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.

1 . A composition comprising compound comprising a polypeptide conjugate, wherein the polypeptide of the polypeptide conjugate comprises:
 i) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80;   ii) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or   iii) a biologically active fragment of i) or ii),   
       and wherein the polypeptide of the compound binds a second polypeptide comprising
 a) an amino acid sequence as provided in any one of SEQ ID NO's 1 to 8; and/or 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 1 to 8. 
 
     
     
         2 . The composition of  claim 1 , wherein the polypeptide comprises an amino acid sequence which is at least 90% identical to any one or more of SEQ ID NO's 48 to 80. 
     
     
         3 . The composition of  claim 1 , wherein the polypeptide is conjugated to a therapeutic agent. 
     
     
         4 . The composition of  claim 3 , wherein the therapeutic agent is an antigen. 
     
     
         5 . The composition of  claim 3 , wherein the therapeutic agent is selected from the group consisting of a cytotoxic agent, a cancer antigen, a self antigen, an allergen, or an antigen from an infectious organism. 
     
     
         6 . The composition of  claim 3 , wherein the therapeutic agent is a drug and/or pharmacological agent. 
     
     
         7 . The composition of  claim 1 , wherein the polypeptide is conjugated to a detectable label. 
     
     
         8 . A composition comprising a compound that binds a polypeptide which comprises:
 i) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80;   ii) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or   iii) a biologically active fragment of i) or ii).   
     
     
         9 . The composition of  claim 8  which binds a polypeptide which comprises an amino acid sequence which is at least 90% identical to any one or more of SEQ ID NO's 48 to 80. 
     
     
         10 . The composition of  claim 8  which is a polypeptide. 
     
     
         11 . The composition of  claim 10  which is an antibody or antigenic binding fragment thereof. 
     
     
         12 . The composition of  claim 11 , wherein the antibody is a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody. 
     
     
         13 . The compound according to  claim 8  which is conjugated to a therapeutic agent or a detectable label. 
     
     
         14 . The composition of  claim 13 , wherein the compound is conjugated to a therapeutic agent. 
     
     
         15 . The composition of  claim 14 , wherein the therapeutic agent is an antigen. 
     
     
         16 . The composition of  claim 13 , wherein the therapeutic agent is a cytotoxic agent, a cancer antigen, a self antigen, an allergen, and/or an antigen from an infectious organism. 
     
     
         17 . The composition of  claim 13 , wherein the therapeutic agent is a drug and/or pharmacological agent. 
     
     
         18 . (canceled) 
     
     
         19 . The composition according to  claim 1  wherein the composition comprises a pharmaceutically acceptable carrier. 
     
     
         20 . The composition of  claim 19  which comprises an adjuvant. 
     
     
         21 . The composition of  claim 19 , wherein the compound is encapsulated in, or exposed on the surface of, a liposome. 
     
     
         22 . A method of modulating an immune response in a subject, the method comprising administering to the subject a composition according to  claim 1 . 
     
     
         23 . The method of  claim 22 , wherein an immune response to an antigen is induced and/or enhanced. 
     
     
         24 . The method of  claim 22 , wherein an immune response to a self antigen or allergen is reduced. 
     
     
         25 . A method of modulating an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to a composition according to  claim 3 , and administering said cells to the subject. 
     
     
         26 . The method of  claim 25 , wherein the cells have been isolated from the subject. 
     
     
         27 .- 28 . (canceled) 
     
     
         29 . A method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject a composition according to  claim 3 , wherein. 
     
     
         30 . (canceled) 
     
     
         31 . A method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject an isolated polynucleotide and/or construct encoding said polynucleotide which, when present in a cell of the subject, modulates the level of activity of a polypeptide which comprises:
 i) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; and/or   ii) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80,   
       in the cell when compared to a cell that lacks said polynucleotide. 
     
     
         32 . The method of  claim 31 , wherein the polynucleotide down-regulates the level of activity of the polypeptide in the cell, and wherein the polynucleotide is selected from: an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, and a double stranded RNA. 
     
     
         33 . The method according to  claim 29 , wherein the disease involving dendritic cells or precursors thereof is cancer, an infection, an autoimmune disease or an allergy. 
     
     
         34 . The method of  claim 33 , wherein the autoimmune disease is lupus erythematosus. 
     
     
         35 . The method of  claim 33 , wherein the infection is a  Plasmodium  sp. infection. 
     
     
         36 . A method of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering
 i) a polypeptide comprising
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or 
 c) a biologically active fragment of a) or b), 
   ii) a composition according to  claim 1 .   
     
     
         37 . A method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering
 i) a polypeptide comprising
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or 
 c) a biologically active fragment of a) or b), 
   ii) a composition according to  claim 1 .   
     
     
         38 . A method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a
 i) a polypeptide comprising
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or 
 c) a biologically active fragment of a) or b), 
   ii) a composition according to  claim 1 .   
     
     
         39 . A method of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, and/or modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells and/or modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising:
 administering to the subject a compound which modulates the production of a polypeptide which comprises:   i) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80;   ii) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or   iii) a biologically active fragment of i) or ii).   
     
     
         40 .- 41 . (canceled) 
     
     
         42 . The method according to  claim 39 , wherein the compound is a polynucleotide. 
     
     
         43 . The method of  claim 42 , wherein the polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in a cell of an animal. 
     
     
         44 . The method of  claim 42 , wherein the polynucleotide down-regulates mRNA levels from a gene encoding the polypeptide. 
     
     
         45 . The method of  claim 44 , wherein the polynucleotide is selected from: an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, and a double stranded RNA. 
     
     
         46 . The method of  claim 42 , wherein the polynucleotide up-regulates mRNA levels from a gene encoding the polypeptide. 
     
     
         47 . The method of  claim 36 , wherein the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is increased. 
     
     
         48 . The method of  claim 36 , wherein the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is decreased. 
     
     
         49 . The method of  claim 37 , wherein antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is increased. 
     
     
         50 . The method of  claim 37 , wherein antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is decreased. 
     
     
         51 . The method of  claim 38 , wherein the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is increased. 
     
     
         52 . The method of  claim 38 , wherein the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, is decreased. 
     
     
         53 . The method according to  claim 36 , wherein the subject is suffering from a disease selected from: graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury. 
     
     
         54 .- 62 . (canceled) 
     
     
         63 . A method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
 i) contacting a cell with the composition of  claim 8 , and   ii) determining whether the polypeptide is present or absent,   
       wherein the presence of the polypeptide provides a diagnosis, prognosis and/or status of the disease. 
     
     
         64 . The method of  63 , wherein the compound is an antibody or antigenic binding fragment thereof. 
     
     
         65 . The method of  claim 63 , wherein the compound is detectably labelled. 
     
     
         66 . The method according to  claim 63  which is performed in vivo on a subject. 
     
     
         67 . The method according to  claim 63  which is performed in vitro on a sample obtained from a subject. 
     
     
         68 . The method according to  claim 63 , wherein the disease is selected from: graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury. 
     
     
         69 . A method of monitoring the effectiveness of a therapy for killing a cell, the method comprising;
 i) exposing a cell to the therapy, and   ii) detecting a cell with a disrupted cell membrane, a dying cell or a dead cell, or a portion thereof, using a method according to  claim 63 ,   
       wherein the presence of a cell with a disrupted cell membrane, a dying cell or a dead cell indicates that the therapy is effective. 
     
     
         70 . The method of  claim 69 , wherein the cell in step i) is in vivo. 
     
     
         71 . The method of  claim 69 , wherein the therapy is administered to a subject. 
     
     
         72 . The method of  claim 69 , wherein the subject has cancer or an infection. 
     
     
         73 . The method according to  claim 69 , wherein step ii) is performed on a sample obtained from a subject. 
     
     
         74 . The method according to  claim 69 , wherein the therapy is drug therapy or radiotherapy. 
     
     
         75 . A method of distinguishing between an early stage apoptotic cell and a late stage apoptotic cell, necrotic cell or dead cell, the method comprising
 i) contacting a cell with a composition according to  claim 1 , and   ii) determining whether binding of the compound to the polypeptide is present or absent, wherein the compound binding to the polypeptide indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.   
     
     
         76 . A method of modulating an immune response to an antigen in a subject, the method comprising
 i) obtaining a population of dendritic cells or precursors thereof,   ii) modulating the production and/or activity of a polypeptide which comprises:
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or 
 c) a biologically active fragment of a) or b), 
   iii) contacting the dendritic cells or precursors thereof with the antigen, and   iv) administering the dendritic cells or precursors thereof to the subject.   
     
     
         77 . The method of  claim 76 , wherein step iii) comprises contacting the dendritic cells or precursors thereof with a cell with a disrupted cell membrane, a cell infected with a pathogen, a dying cell, a dead cell, and/or a portion thereof, comprising said antigen. 
     
     
         78 . The method of  claim 76 , wherein step iii) comprises
 a) obtaining a cell comprising the antigen,   b) disrupting the cell membrane of the cell, and   c) contacting the product of step b) with the dendritic cells or precursors thereof.   
     
     
         79 . The method according to  claim 76  which further comprises, before step ii), enriching the population for cells expressing the polypeptide. 
     
     
         80 . A method of enriching dendritic cells, or a subset or precursors thereof, from a sample comprising;
 i) contacting a sample comprising dendritic cells or precursors thereof with a compound according to  claim 1 , and   ii) isolating cells bound to the compound.   
     
     
         81 . A method of enriching dendritic cells, or a subset or precursors thereof, from a sample comprising;
 i) contacting a sample comprising dendritic cells or precursors thereof with a detectably labelled first polynucleotide that hybridizes to a second polynucleotide encoding a polypeptide which comprises
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; and/or 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80, and 
   ii) isolating the detectably labelled cells.   
     
     
         82 . The method of  claim 80 , wherein the cells obtained from step ii) are administered to a subject. 
     
     
         83 . The method of  claim 82 , wherein the cells are administered to treat and/or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy. 
     
     
         84 . A method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising;
 i) contacting a sample comprising dendritic cells or precursors thereof with a compound according to  claim 1 , and   ii) detecting cells bound to the compound.   
     
     
         85 . A method of detecting dendritic cells, or a subset or precursor thereof, in a sample comprising;
 i) contacting a sample comprising dendritic cells or precursors thereof with a detectably labelled first polynucleotide that hybridizes to a second polynucleotide encoding a polypeptide which comprises
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; and/or 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80, and 
   ii) detecting the detectably labelled cells.   
     
     
         86 . A method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising;
 i) administering to the subject a compound according to  claim 1 ,   ii) detecting cells bound to the compound.   
     
     
         87 . The method of  claim 84 , wherein the compound is detectably labelled. 
     
     
         88 . A method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising;
 i) administering to the subject a detectably labelled first polynucleotide that hybridizes to a second polynucleotide encoding a polypeptide which comprises
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; and/or 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80, and, 
   ii) detecting the detectably labelled cells.   
     
     
         89 . The method according to  claim 80 , wherein the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CD11c, HLADR and BDCA3. 
     
     
         90 . A method of detecting a cell with a disrupted cell membrane, a cell infected with a pathogen, a dying cell or a dead cell, the method comprising
 i) contacting a cell with a compound that binds a polypeptide comprising
 a) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80; 
 b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or 
 c) a biologically active fragment of a) or b), and 
   ii) determining whether binding of the compound to the polypeptide is present or absent, wherein the compound binding to the polypeptide indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying or is dead.   
     
     
         91 . An isolated and/or exogenous polynucleotide encoding a compound according to  claim 1 , wherein the compound is a polypeptide. 
     
     
         92 . An isolated polynucleotide which, when present in a cell of a subject, modulates the level of activity of a polypeptide in the cell when compared to a cell that lacks said polynucleotide, wherein the polypeptide comprises
 i) an amino acid sequence as provided in any one of SEQ ID NO's 48 to 80;   ii) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NO's 48 to 80; and/or   iii) a biologically active fragment of i) or ii).   
     
     
         93 . A vector comprising a polynucleotide of  claim 91 . 
     
     
         94 . The vector of  claim 93  which is an expression vector. 
     
     
         95 . A host cell comprising at least one polynucleotide of  claim 91 . 
     
     
         96 . The host cell of  claim 95  which is a bacterial, yeast, insect, animal or plant cell. 
     
     
         97 . An enriched population of dendritic cells and/or precursors thereof, obtained by a method according to any  claim 80 . 
     
     
         98 . An expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof according to  claim 97 . 
     
     
         99 . A composition comprising a polynucleotide of  claim 91 , and a pharmaceutically acceptable carrier. 
     
     
         100 . A kit comprising a composition according to  claim 1 .